CN112546228A

CN112546228A - Use of non-IGF 1R-binding substances for preventing and/or treating inflammatory diseases

Info

Publication number: CN112546228A
Application number: CN201910854478.3A
Authority: CN
Inventors: 时玉舫; 王莹; 王雪枫
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2021-03-26
Also published as: US20220401515A1; WO2021047607A1; EP4046658A4; CN113164606A; EP4046658A1; AU2020346948A1

Abstract

The invention provides application of a non-IGF 1R-bound substance in preventing and/or treating inflammatory diseases, and particularly provides application of a non-IGF 1R-bound substance in preparing a composition or a preparation, wherein the composition or the preparation is used for preventing and/or treating inflammatory diseases.

Description

Use of non-IGF 1R-binding substances for preventing and/or treating inflammatory diseases

Technical Field

The invention relates to the field of biomedicine, in particular to application of a non-IGF 1R-bound substance in preventing and/or treating inflammatory diseases.

Background

Insulin-like growth factor 2(insulin like growth factor2, IGF2) belongs to the insulin-like growth factor family, and is involved in regulating cell proliferation, differentiation, metabolism, aging and death together with other families, affecting the development, growth, health and disease of organisms^1,2. IGF2 receptors include IGF1R, IGF2R, which has the highest affinity for IGF2R and, secondly, may bind to IGF2RIGF1R binds with little to insulin receptor binding²。

It is now believed that the major biological functions of IGF2 are dependent on activation of the tyrosine kinase of IGF1R without a positive association with IGF2R and IR²。

About 90% of IGF2R is distributed in the intracellular Golgi apparatus, and the remaining 10% of IGF2R is distributed in the extracellular membrane³. IGF2R is not the only receptor for IGF2, IGF2 is not the only ligand for IGF2R, and its ligand also includes mannose-6-phosphate, and in the prior reports, the main function of IGF2R is to transport its ligand to lysosomes for protein processing or degradation⁴. In addition, it has been found that IGF2R on the cell membrane can be cleaved off in a soluble form, enter the blood and other fluid environments, effectively bind IGF2, and inhibit the function of IGF2^4,5。

Autoimmune diseases are diseases caused by abnormal attack of the immune system on self organs and tissue cells, and data published by the U.S. department of health and human services show that 2400 ten thousand americans at present, up to 7% of the U.S. population suffer from more than eighty autoimmune diseases, and almost all parts of the body are involved^6,7. The pathogenic factors of most autoimmune diseases are not clear, and may be related to genetic factors, infection factors, drugs, environment factors and the like, the disease course is long, repeated remission and attack occur, and women are more than men in the patients with the disease. The majority of autoimmune diseases involve the involvement of monocytes and macrophages.

In various autoimmune diseases, activated macrophages secrete Milk fat globule-epidermal growth factor 8 (Milk fat globule-epidermal growth factor 8, MFG-E8), the C-terminal structural domain of MFG-E8 is combined with phosphatidylserine of apoptotic cells, the phagocytosis of apoptotic cells by macrophages is mediated, cells which are attacked and apoptotic in autoimmune diseases and dead immune cell fragments are eliminated, and the body is helped to recover homeostasis⁸. Some inflammatory factors, such as lipopolysaccharide, can inhibit macrophage expression of MFG-E8, inhibit apoptosis debris clearance and aggravate inflammatory response⁹. In contrast, MFG-E8-mediated macrophagyThe cell phagocytosis of apoptosis debris can inhibit the inflammation promoting effect of inflammatory factors such as lipopolysaccharide on macrophage, including inhibition of p38, ERK1/2, c-JNK, MAPK and p65¹⁰。

Inflammation is the body's defense against the production of inflammatory factors, both intrinsic and extrinsic, including biological, physical, chemical, foreign, necrotic, allergic and pathological changes, and is regulated by inflammatory factors and the body itself¹¹. In inflammatory diseases, the innate immunity and the adaptive immunity are involved, and in the early stage of inflammation, the innate immunity firstly reaches the tissue injury part to influence and determine the response types of the innate immunity and the adaptive immunity in the later stage¹². When the inflammatory regulation is disturbed or disturbed, i.e. inflammatory diseases occur, the ability to resist stimuli and pathogens is lost, and the disease condition is accelerated. The inflammatory response includes both acute and chronic inflammatory responses, in terms of the rate of inflammatory response.

There are two major classes of current drugs for the treatment of inflammatory and autoimmune diseases: the first is steroid anti-inflammatory drug, i.e. steroid drug, such as adrenocortical hormone, androgen and estrogen, which has certain anti-inflammatory effect and will cause endocrine disturbance and other multi-system dysfunction after long-term use^13-15. The second class of non-steroidal anti-inflammatory drugs comprises aspirin, acetaminophen, indomethacin, naproxen, naproxone, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib and the like, and is widely used for anti-inflammatory treatment of osteoarthritis, rheumatoid arthritis and the like in clinic^14-16. The nonsteroidal anti-inflammatory drug has single action principle, and mainly plays an anti-inflammatory role by inhibiting the synthesis of prostaglandin, reducing the formation of bradykinin and reducing the aggregation and aggregation of white blood cells and platelets¹⁷. Therefore, the steroid drugs are limited by side effects, the non-steroid drugs are limited by single action path, and the sensitization effect of the combination drugs is poor.

Therefore, there is an urgent need in the art to develop a drug that can more effectively treat autoimmune and/or inflammatory diseases.

Disclosure of Invention

The invention aims to provide a medicament capable of treating autoimmune diseases and/or inflammatory diseases more effectively.

The invention provides the use of a substance other than IGF 1R-bound form for the preparation of a composition or formulation for the prevention and/or treatment of an inflammatory disease.

In another preferred embodiment, the non-IGF 1R-binding form is selected from the group consisting of: an IGF2 mutant, a vector expressing an IGF2 mutant, an antibody, a small molecule compound, or a combination thereof.

In another preferred embodiment, the inflammatory disease is selected from the group consisting of: peritonitis, inflammatory bowel disease, multiple sclerosis, diabetes, systemic lupus erythematosus, scleroderma, hashimoto's thyroiditis, autoimmune hepatitis, autoimmune uveitis, interstitial lung disease, psoriasis, vitiligo, dermatomyositis, kawasaki disease, adult otopathy, ankylosing spondylitis, sarcoidosis, arthritis associated with onset and stop inflammation, polyarticular juvenile idiopathic arthritis, rheumatoid arthritis, graft-versus-host disease, autoimmune pancreatitis, parkinson's disease, senile dementia, or a combination thereof.

In another preferred embodiment, the non-IGF 1R-binding substances include substances that are selective or have a higher affinity for IGF2R than for IGF1R, and substances that do not activate IGF1R (e.g., non-IGF 1R ligands such as retinoic acid) that are highly active for IGF 2R.

In another preferred embodiment, the vector expressing the IGF2 mutant comprises a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of: an adenoviral vector, a lentiviral vector, or a combination thereof.

In another preferred embodiment, the non-IGF 1R-binding substance further comprises one or more substances selected from the group consisting of: IGF2R, such as Plasminogen (Plasminogen), filaggrin (Serglycin), cell inhibitor of E1a stimulatory gene (CREG), Sulfamidase (Sgsh), Phosphorylated β -glucuronidase (GUSb), effectively inhibits inflammatory ligands.

In another preferred example, the IGF2 mutant is mutated at the tyrosine corresponding to position 27 of SEQ ID No. 1 of wild-type IGF2 protein.

In another preferred embodiment, the tyrosine mutation at position 27 is one or more amino acids selected from the group consisting of: leucine, isoleucine, valine, methionine, alanine, phenylalanine, serine, proline, threonine, histidine, lysine, tryptophan, arginine, glutamic acid, glycine, aspartic acid, cysteine.

In another preferred embodiment, the tyrosine at position 27 is mutated to leucine.

In another preferred example, the IGF2 mutant is mutated in the wild-type IGF2 protein corresponding to glutamic acid at position 12 of SEQ ID No.:1, and optionally deletes D-domain (threonine 62 to glutamic acid 67 of SEQ ID No.: 1).

In another preferred embodiment, the glutamic acid mutation at position 12 is one or more amino acids selected from the group consisting of:

Asp、Ala、Gln、His、Arg、Lys。

in another preferred example, the IGF2 mutant is mutated in the wild-type IGF2 protein with respect to the phenylalanine at position 26 of SEQ ID No.:1 and optionally deletes D-domain (threonine 62 to glutamic acid 67 of SEQ ID No.: 1).

In another preferred embodiment, the phenylalanine at position 26 is mutated to one or more amino acids selected from the group consisting of: ser, Asp, Ala, Gln, His, Arg, Lys.

In another preferred embodiment, the phenylalanine at position 26 is mutated to serine.

In another preferred example, the IGF2 mutant is mutated in the wild-type IGF2 protein in the tyrosine corresponding to position 27 of SEQ ID No.:1 and optionally deleting D-domain (threonine 62 to glutamic acid 67 of SEQ ID No.: 1).

In another preferred embodiment, the tyrosine mutation at position 27 is one or more amino acids selected from the group consisting of: leu, Asp, Ala, Gln, His, Arg, Lys.

In another preferred example, the IGF2 mutant is mutated in the wild-type IGF2 protein corresponding to valine at position 43 of SEQ ID No.:1, and optionally deletes D-domain (threonine 62 to glutamic acid 67 of SEQ ID No.: 1).

In another preferred embodiment, the valine at position 43 is mutated to one or more amino acids selected from the group consisting of: leu, Asp, Ala, Gln, His, Arg, Lys.

In another preferred embodiment, the valine at position 43 is mutated to leucine.

In another preferred embodiment, the tyrosine mutation at position 27 is one or more amino acids selected from the group consisting of: leu, Asp, Ala, Gln, His, Arg, Lys, and simultaneously mutating valine at position 43 to one or more amino acids selected from the group consisting of: leu, Asp, Ala, Gln, His, Arg, Lys.

In another preferred embodiment, the tyrosine at position 27 and the valine at position 43 are mutated to leucine simultaneously.

In another preferred embodiment, the tyrosine at position 27 and the valine at position 43 are simultaneously mutated to leucine and optionally deleted for D-domain (threonine 62 to glutamic acid 67 of SEQ ID No.: 1).

In another preferred embodiment, the amino acid sequence of the IGF2 mutant is shown in any one of SEQ ID NO. 2-58.

In another preferred embodiment, the IGF2 mutant is a polypeptide having an amino acid sequence as shown in any one of SEQ ID Nos. 2-58, an active fragment thereof, or a conservatively variant polypeptide thereof.

In another preferred embodiment, the IGF2 mutant has an amino acid sequence identical or substantially identical to the sequence shown in SEQ ID No. 1 except for the mutations (e.g., Glu at 12, Phe at 26, Tyr at 27, Val at 43, and the corresponding mutant sequence for deletion of D-domain).

In another preferred embodiment, the glutamic acid at position 12 is mutated to glutamine.

In another preferred embodiment, the substantial identity is a difference of at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids, wherein the difference comprises amino acid substitution, deletion or addition (e.g., deletion of T at position 62 to E at position 67), and the IGF2 mutant has an activity of inhibiting inflammation.

In another preferred embodiment, the homology to the sequence as shown in SEQ ID No. 1 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, most preferably at least 98% or 99%.

In another preferred embodiment, the IGF2 protein is derived from a human or non-human mammal.

In another preferred embodiment, the IGF2 mutant is selected from the group consisting of:

(a) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID No. 2-58;

(b) a polypeptide which is formed by substituting, deleting or adding one or more (such as 2, 3, 4 or 5) amino acid residues in the amino acid sequence shown in any one of SEQ ID NO. 2-58, and has the inflammation inhibiting activity and is derived from the (a).

In another preferred embodiment, the derived polypeptide has at least 60%, preferably at least 70%, more preferably at least 80%, most preferably at least 90%, such as 95%, 97%, 99% homology with the sequence as shown in any of SEQ ID No. 2-58.

In another preferred example, the IGF2 mutant is formed by mutating a wild-type IGF2 protein shown in SEQ ID NO. 1.

In another preferred embodiment, the composition or formulation is further used for one or more uses selected from the group consisting of:

(i) reducing pathogenic mononuclear cell infiltration in the colon;

(ii) inhibiting the pathogenic phenotype of macrophages;

(iii) reducing macrophage infiltration number;

(iv) immune memory that confers significant inhibition of inflammation to macrophages;

(v) preventing and/or treating autoimmune diseases;

(vi) the macrophage-involved diseases (such as inflammatory reaction related to the occurrence of diseases such as hepatic fibrosis, renal fibrosis and pulmonary fibrosis, inflammatory reaction caused by tissue, organ or cell transplantation, inflammatory reaction related to the occurrence and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma and lymphoma, and inflammatory reaction related to autoimmune diseases);

(vii) facilitating the implantation of the transplanted tissue or cells.

In another preferred embodiment, the autoimmune disease is selected from the group consisting of: multiple sclerosis, inflammatory bowel disease, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, insulin resistance, diabetes, autoimmune hepatitis, vitiligo, psoriasis, peritonitis, scleroderma, hashimoto's thyroiditis, graft versus host disease, dermatomyositis, kawasaki disease, adult otopathy, ankylosing spondylitis, sarcoidosis, arthritis associated with onset and stop inflammation, polyarticular juvenile idiopathic arthritis, autoimmune pancreatitis, and in part undefined autoimmune disease, or a combination thereof.

In another preferred embodiment, the composition comprises a pharmaceutical composition.

In another preferred embodiment, the composition comprises (a) a substance that is not IGF 1R-binding; and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the composition comprises from 0.001 to 99 wt%, preferably from 0.1 to 90 wt%, more preferably from 1 to 80 wt% of a non-IGF 1R-bound material, based on the total weight of the composition.

In another preferred embodiment, the composition is a liquid preparation or a freeze-dried preparation.

In another preferred embodiment, the composition is an injection.

In a second aspect, the invention provides a cell preparation comprising:

macrophages treated with a non-IGF 1R-binding agent.

In another preferred embodiment, the non-IGF 1R-binding form is selected from the group consisting of: IGF2, IGF2 mutants, vectors expressing IGF2 mutants, antibodies, small molecule compounds, or combinations thereof.

In another preferred embodiment, the cell preparation further comprises a substance that is not IGF 1R-binding.

In another preferred embodiment, the macrophage has a characteristic selected from the group consisting of:

(a) IGF1R inactivated, IGF2R activated; and/or

(b) Macrophages phosphorylate with oxidation to the primary capacitation; and/or

(c) Having one or more characteristics selected from the group consisting of:

low expression of IL-1 β;

low expression of TNF α;

low expression of CXCL 10;

high expression of CCL 22;

high expression of CLEC7 a;

high expression PD-L1;

high expression PD-L2;

high expression of IL-10;

high expression of TGF-. beta.s.

In another preferred embodiment, the macrophages are derived from bone marrow, abdominal cavity, peripheral blood, site of inflammation occurrence, or a combination thereof.

In another preferred embodiment, the cell preparation comprises a liquid preparation.

In another preferred embodiment, the cells in the cell preparation consist essentially (. gtoreq.90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or entirely of (a) macrophages pre-treated with a substance that is not IGF 1R-binding type and (b) optionally a substance that is not IGF 1R-binding type.

In another preferred embodiment, the concentration of said macrophages in said composition is 1X 10⁴－5×10⁷Per ml, preferably 5X 10⁴－5×10⁶Per ml, more preferably 1X 10⁵－1×10⁶/ml。

In another preferred embodiment, the carrier is selected from the group consisting of: an infusion solution carrier and/or an injection carrier, preferably, the carrier is one or more selected from the following group: normal saline, dextrose saline, or combinations thereof.

In another preferred embodiment, the cell preparation further comprises other drugs for preventing and/or treating inflammatory diseases.

In another preferred embodiment, the other agent for preventing and/or treating inflammatory diseases is selected from the group consisting of: non-steroidal anti-inflammatory drugs, glucocorticoids, methotrexate, TNF α neutralizing antibodies, TNFR1 antibodies, TNFR2 antibodies, anti-CD 20 antibodies, IL-1R antagonists, IL-12 and IL-23p40 neutralizing antibodies, IL-23p19 neutralizing antibodies, IL-17A receptor neutralizing antibodies, or combinations thereof.

In another preferred embodiment, the additional agent for preventing and/or treating autoimmune diseases is selected from the group consisting of: non-steroidal anti-inflammatory drugs, glucocorticoids, methotrexate, TNF α neutralizing antibodies, TNFR1 antibodies, TNFR2 antibodies, anti-CD 20 antibodies, IL-1R antagonists, IL-12 and IL-23p40 neutralizing antibodies, IL-23p19 neutralizing antibodies, IL-17A receptor neutralizing antibodies, CTLA-4 fusion proteins, or combinations thereof.

In a third aspect the invention provides a kit comprising:

(i) a first container, and contained in the first container, activated macrophages in which the active ingredient (a) has been treated with a substance other than IGF 1R-bound type, or a drug containing the active ingredient (a);

(ii) optionally a second container, and contained therein an active ingredient (b) other than IGF 1R-bound form, or a medicament containing the active ingredient (b); and

(iii) the specification describes a description of the combined administration of active ingredient (a) and active ingredient (b) for the prevention and/or treatment of inflammatory diseases.

In another preferred embodiment, the kit further comprises:

(iv) a third container, and the active ingredient (c) contained in the third container, or a medicament containing the active ingredient (c) as an additional agent for preventing and/or treating an inflammatory disease.

In another preferred embodiment, the kit further comprises:

(v) a fourth container, and the active ingredient (d) contained in the fourth container is other medicines for preventing and/or treating autoimmune diseases or medicines containing other medicines for preventing and/or treating autoimmune diseases.

In another preferred embodiment, the first container, the second container, the optional third container and the optional fourth container may be the same or different.

In another preferred embodiment, the drug in the first container is a single formulation comprising macrophages treated with a substance other than IGF1R conjugated.

In another preferred embodiment, the drug in the second container is a single formulation containing a non-IGF 1R conjugated type of substance.

In another preferred embodiment, the third container is a single preparation containing other drugs for preventing and/or treating inflammatory diseases.

In another preferred embodiment, the fourth container is a single preparation containing other drugs for preventing and/or treating autoimmune diseases.

In another preferred embodiment, the dosage form of the drug is an injectable dosage form.

In another preferred embodiment, the kit further comprises instructions describing the combined administration of active ingredient (a), active ingredient (b), optionally (c) and optionally (d) to (i) prevent and/or treat an inflammatory disease; and/or (ii) instructions for the prevention and/or treatment of an autoimmune disease.

In another preferred embodiment, the concentration of the non-IGF 1R-binding substance in the formulation (b) containing a non-IGF 1R-binding substance is 0.01. mu.g-1 mg/kg body weight, preferably 0.1-10. mu.g/kg body weight, more preferably 0.5-5. mu.g/kg body weight.

In another preferred embodiment, the macrophage concentration in the preparation containing (a) macrophages treated with a substance other than IGF 1R-binding type is 0.2 x 10⁴-1*10⁸One/kg body weight, preferably 1 x 10⁵-5*10⁷One/kg body weight, more preferably 1 x 10⁶-5*10⁶One per kg body weight.

In another preferred embodiment, in the preparation containing (c) the other agent for preventing and/or treating inflammatory diseases, the concentration of the other agent for preventing and/or treating inflammatory diseases is 0.0002 to 40mg/kg body weight, preferably 0.001 to 30mg/kg body weight, more preferably 0.02 to 25mg/kg body weight.

In another preferred embodiment, the concentration of the other agent for preventing and/or treating autoimmune diseases in the preparation containing (d) the other agent for preventing and/or treating autoimmune diseases is 0.0002 to 0.1mg/kg body weight, preferably 0.001 to 0.08mg/kg body weight, more preferably 0.002 to 0.01mg/kg body weight.

The fourth aspect of the invention provides a medicine sieving method, which comprises the following steps:

(a) in the test group, adding a test substance to a culture system of cells, and observing the binding activity of the test substance to IGF1R and/or IGF2R in the cells of the test group; in the control group, the test substance was not added to the culture system of the same cells;

wherein if the binding activity of the test agent to IGF1R is reduced in the cells of the test group; and the binding activity to IGF2R did not change or increased, indicating that the test substance is a non-IGF 1R-bound version of IGF 2.

In another preferred example, the method further comprises the steps of:

(b) further determining the binding activity of the non-IGF 1R-bound IGF2 mutant obtained in step (a) to an IGFBP protein; and/or further tested for unchanged or slightly increased binding to IGFBP proteins.

In another preferred example, the method further comprises the steps of:

(c) introducing a positive control, which is an IGF2 protein or IGF2 mutant, and comparing its binding activity to IGF1R and/or IGF2R with the binding activity of the non-IGF 1R-binding IGF2 mutant obtained in step (a) to IGF1R and/or IGF 2R.

In another preferred example, the binding activity of the non-IGF 1R-binding form of the mutant IGF2 obtained in step (a) for IGF1R and/or IGF2R is comparable to the binding activity of the positive control for IGF1R and/or IGF 2R.

The fifth aspect of the invention provides a medicine sieving method, which comprises the following steps:

(a) in the test group, adding a test substance to a culture system of cells, and observing the effect of the test substance on the binding activity of IGF2 with IGF1R and/or IGF2R in the cells of the test group; in the control group, the test substance was not added to the culture system of the same cells;

wherein if the test substance inhibits the binding activity of IGF2 to IGF1R in the cells of the test group; and has no effect or promotion effect on the binding activity of IGF2 to IGF2R, it is suggested that the test substance is a non-IGF 1R-binding type substance.

In another preferred embodiment, the non-IGF 1R-binding form is selected from the group consisting of: an antibody, a small molecule compound, or a combination thereof.

In another preferred example, the method further comprises the steps of:

(b) further determining the binding activity of the non-IGF 1R-bound material obtained in step (a) to an IGFBP protein; and/or further tested for unchanged or slightly increased binding to IGFBP proteins.

In another preferred example, the method further comprises the steps of:

(c) introducing a positive control, which is an IGF2 protein or IGF2 mutant, and comparing its binding activity to IGF1R and/or IGF2R with the binding activity of the non-IGF 1R-bound material obtained in step (a) to IGF1R and/or IGF 2R.

In another preferred example, the binding activity of the non-IGF 1R-binding form of the substance obtained in step (a) on IGF1R and/or IGF2R is comparable to the binding activity of the positive control on IGF1R and/or IGF 2R.

In a sixth aspect, the present invention provides a method for obtaining a proinflammatory sample and an anti-inflammatory sample in vitro, comprising:

in a first set of experiments, a low dose of IGF2 was used for treatment to obtain an anti-inflammatory sample; and

in a second set of experiments, pro-inflammatory samples were obtained by treatment with high doses of IGF 2.

In another preferred example, the binding activity of IGF2 to IGF2R is further determined in the proinflammatory sample.

In another preferred embodiment, the binding activity of IGF2 on IGF2R is further determined in the inflammation-suppressive sample.

The seventh aspect of the present invention provides a composition comprising:

(a) a non-IGF 1R-bound IGF2 mutant obtained by the method of the fourth aspect of the invention; wherein the non-IGF 1R-binding IGF2 mutant does not comprise an IGF2 mutant selected from the group consisting of: wild-type IGF2, 25-91 amino acid fragments of wild-type IGF2, IGF2 mutant in which glutamic acid at position 12 is mutated to Asp, Ala, Gln, His, Arg, or Lys, IGF2 mutant in which phenylalanine at position 26 is mutated to Ser, IGF2 mutant in which tyrosine at position 27 is mutated to Leu, IGF2 mutant in which valine at position 43 is mutated to Leu, IGF2 mutant in which D-domain is deleted, or a combination thereof.

In another preferred example, the non-IGF 1R-binding form of mutant IGF2 does not include the mutant IGF2 shown in any of SEQ ID nos 1-2, 16, 18, 20, 22, 24, 26, 28, 43.

In another preferred embodiment, the amino acid sequence of the non-IGF 2 conjugated form of IGF1R is as set forth in any one of SEQ ID NO. 3-15, 17, 19, 21, 23, 25, 27, 29-42, 44-58.

In an eighth aspect, the present invention provides a method of inhibiting inflammatory activity comprising the steps of:

culturing macrophages in the presence of a non-IGF 1R-bound agent, thereby inhibiting inflammatory activity.

In another preferred embodiment, the non-IGF 1R binding form is administered at a concentration of 0.0000005-0.0005 mg/ml, preferably 0.000001-0.0001mg/ml, more preferably 0.000005-0.00005 mg/ml.

In a ninth aspect, the present invention provides a method for preventing and/or treating inflammatory diseases, comprising:

(a) administering to a subject in need thereof a cell preparation according to the second aspect of the invention or a kit according to the third aspect of the invention or a composition according to the seventh aspect of the invention.

In another preferred embodiment, the subject includes non-human mammals and humans.

In another preferred embodiment, the non-IGF 1R-binding form is administered in a dose of 0.01 μ g to 1mg/kg body weight, preferably 0.1 μ g to 10 μ g/kg body weight, more preferably 0.5 μ g to 5 μ g/kg body weight.

In another preferred embodiment, the non-IGF 1R-binding form of the substance is administered at a frequency of 1 to 2 times per day.

In another preferred embodiment, said administering comprises simultaneous administration or sequential administration.

In a tenth aspect, the present invention provides a method for preventing and/or treating an autoimmune disease, comprising:

In an eleventh aspect, the invention provides a use of the cell preparation of the second aspect, the kit of the third aspect or the composition of the seventh aspect, for the preparation of a medicament for the prevention and/or treatment of an inflammatory disease.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows that low dose IGF2 inhibited peritonitis. Peritonitis mice were treated with low-dose IGF2(L-IGF2, 5-50ng IGF2 per mouse) and high-dose IGF2(H-IGF2, 1000ng IGF2 per mouse), respectively. Peritoneal monocytes and macrophages were isolated from peritonitis mice and analyzed for the number of peritoneal macrophages that could be isolated from each mouse. The experiment was repeated three times, with significant differences being tested by student's t. P < 0.05; p < 0.01; p < 0.001.

Figure 2 shows that low dose of IGF2 alters macrophage energy metabolism preference and inflammatory factor expression trends and potentials. Peritonitis mice were treated with low-dose IGF2(L-IGF2, 5-50ng IGF2 per mouse) and high-dose IGF2(H-IGF2, 1000ng IGF2 per mouse), respectively. Peritoneal monocytes and macrophages were isolated and analyzed from peritonitis mice. A-B, low dose IGF2 enhanced macrophage oxidative phosphorylation potential, while high dose IGF2 was ineffective. C, low dose IGF2 decreased macrophage lactate production, corresponding to weaker aerobic glycolytic metabolism. D-F, a low dose of IGF2, inhibits the potential for expression of pro-inflammatory genes, increases the potential for anti-inflammatory repair of related gene expression. The experiment was repeated three times, with significant differences being tested by student's t. P < 0.05; p < 0.01; p < 0.001.

FIG. 3 shows the construction of igf2r gene deficient mice, as well as igf1r or igf2r conditional knockout mice. A-C, using Crisper-Cas9 technology, knocking down IGF2R gene expression (complete knockout of IGF2R gene and death of mouse embryo) by causing frameshift mutation caused by 2bp or 13bp gene deletion, using gene sequencing technology to identify progeny mice, and using flow cytometry to identify knocking-down efficiency. D-I by hybridization of IGF1R^fl/flOr IGF2R^fl/flMouse and Lyz^CreMouse, obtaining specific knockdownMice with myeloablative lymphocytes IGF1R or IGF 2R. The knockout efficiency was identified by PCR and flow analysis. E, agarose gel electrophoresis results, lane 1 indicates wild type mouse (124bp), lane 2 indicates Igf1r^fl/fl(220 bp), no band in lane 3, Igf1r^fl/flMice did not possess Lyz2^CreActivity, Lane 4(320bp) indicates Igf1r^fl/flThe mouse is provided with Lyz2^CreAnd (4) vitality. H, lane 1 indicates wild type mice (2000bp), lane 2Igf 2r^fl/flMouse, without Lyz2^CreViability (2200 bp).

Figure 4 shows that low dose IGF2 inhibits Dextran Sodium Sulfate (DSS) -induced inflammatory bowel disease. Enteritis in the mouse model was induced by feeding 4% DSS solution. a-E, low dose IGF2, ameliorated DSS-induced inflammatory bowel disease, with marked improvement in body mass index, survival time, stool score and stool bleeding score, and colon length. F-H, IGF2, H & E staining of colon tissue, TUNEL and Ki67 immunohistochemical staining of mice in DSS-induced colitis when used. The experiment was repeated three times, with significant differences being tested by student's t. P < 0.05; p < 0.01; p < 0.001.

Figure 5 shows that low dose IGF 2-treated macrophages were effective in inhibiting inflammatory bowel disease. A mouse model of inflammatory bowel disease was induced by feeding 4% DSS solution. a-B, low or high dose IGF 2-induced therapeutic effect of peritoneal macrophages in DSS-induced inflammatory bowel disease, weight change and survival curves were recorded. The experiment was repeated three times, with significant differences being tested by student's t. P < 0.01.

Figure 6 shows that low dose IGF2 reduced pathogenic mononuclear cell infiltration in the colon. A mouse model of inflammatory bowel disease was induced by feeding 4% DSS solution. A, low dose IGF2 ameliorates DSS-induced inflammatory bowel disease, reducing mononuclear cell infiltration in colon tissue. B-C, the effect of IGF2 on the inflammatory phenotype of macrophages infiltrated in the colon of inflammatory bowel disease mice was analyzed by comparing the proportion of interleukin-1 β positive macrophages. The experiment was repeated three times, with significant differences being tested by student's t. P < 0.001.

Figure 7 shows that IGF2R performs the anti-inflammatory task of low-dose IGF 2. Enteritis in the mouse model was induced by feeding 4% DSS solution. Evaluation of body Mass index Low dose IGF2 treatment of DSS in IGF2R^fl/flLyz2^CreMouse and IGF2R^fl/flInflammatory bowel disease induced in mice. B, evaluation of body Mass index high dose IGF2 treatment of DSS in IGF1R^fl/flLyz2^CreMouse and IGF1R^fl/flInflammatory bowel disease induced in mice. The experiment was repeated three times, with significant differences being tested by student's t. P<0.05；***P<0.001。

FIG. 8 shows that Leu27-IGF2 targeted activation of IGF2R inhibits peritonitis and inflammatory bowel disease most effectively. Peritonitis mice were treated with low dose IGF2/Leu27-IGF2(50ng IGF2/Leu27-IGF2 per mouse) and high dose IGF2/Leu27-IGF2(1000ng, or 2000ng IGF2/Leu27-IGF2 per mouse), respectively. Peritoneal monocytes and macrophages were isolated and analyzed from peritonitis mice. A, Leu27-IGF2 preprogrammed relative pH of the cytoplasm of peritoneal macrophages. B-C, ability of peritoneal macrophages preprogrammed with different doses of IGF2 and Leu27-IGF2 to produce nitric oxide and lactic acid. A mouse model of inflammatory bowel disease was induced by feeding 4% DSS. D, body mass index to evaluate the relieving effect of Leu27-IGF2 on DSS-induced inflammatory bowel disease. E, colon Length assessment of the relieving effect of Leu27-IGF2 on DSS-induced inflammatory bowel disease. F, effective threshold schematic for wild-type IGF2 and Leu27-IGF2 in alleviating Ftg-induced peritonitis and DSS-induced inflammatory bowel disease. The experiment was repeated three times, with significant differences being tested by student's t. P < 0.01; p < 0.001. ns, non-significant difference.

Detailed Description

As a result of extensive and intensive studies, the present inventors have for the first time unexpectedly found that a substance other than IGF 1R-bound type can significantly (a) prevent and/or treat inflammatory diseases; and/or (b) preventing and/or treating autoimmune disease. On this basis, the present inventors have completed the present invention.

As used herein, wild-type IGF2, 25-91 amino acid fragments of wild-type IGF2, IGF2 mutant in which glutamic acid at position 12 is mutated to Asp, Ala, gin, His, Arg, or Lys, IGF2 mutant in which phenylalanine at position 26 is mutated to Ser, IGF2 mutant in which tyrosine at position 27 is mutated to Leu, IGF2 mutant in which valine at position 43 is mutated to Leu, and IGF2 mutant in which D-domain is deleted are all amino acid truncations, mutations, and/or deletions performed on the basis of SEQ ID No. 1.

IGF2 protein and mutant thereof

Insulin-like growth factor 2(insulin like growth factor2, IGF2) belongs to the insulin-like growth factor family, and is involved in regulating cell proliferation, differentiation, metabolism, aging and death together with other families, affecting the development, growth, health and disease of organisms^1,2. IGF2 receptors include IGF1R, IGF2R, which has the highest affinity for IGF2R, and secondly binds IGF1R and hardly binds to the insulin receptor²。

IGF2 has high amino acid sequence homology with IGF1, and has 62% overlapping sequence, therefore, IGF2 can often show the biological effect activated by IGF1². It is currently believed that the major biological function of IGF2 is dependent on the activation of the tyrosine kinase of IGF1R for opening, and that there is no positive association with IGF2R and IR. From an evolutionary point of view, IGF1R has genetic homology to IR, and IGF1R is quite similar to the signaling pathway activated downstream of IR. However, the IGF2R gene is completely different from the IGF1R or IR gene in origin, and thus, IGF2R is often considered to have opposite biological functions to IGF1R and IR, and IGF2R acts as a degradation receptor for IGF2 and inhibits the activation of IGF2 on IGF1R by internalistically degrading IGF 2.

About 90% of IGF2R is distributed in the intracellular Golgi apparatus, and the remaining 10% of IGF2R is distributed in the extracellular membrane³. IGF2R is not the only receptor for IGF2, IGF2 is not the only ligand for IGF2R, and its ligand also includes mannose-6-phosphate, and in the prior reports, the main function of IGF2R is to transport its ligand to lysosomes for protein processing or degradation⁴. In addition, IGF2R on the cell membrane can be sheared off in a soluble form into the blood and other fluid environments, effectively binding IGF2 and inhibiting IGF2 from functioning^4,5. Earlier studies of the present invention found IGF2 can inhibit animal models of diseases such as multiple sclerosis and ulcerative colitis, but the specific regulation mechanism is still unclear.

Specifically, the invention relates to IGF2 protein and variants thereof, and in a preferred embodiment of the invention, the amino acid sequence of the IGF2 protein is shown as SEQ ID No. 1. The IGF2 protein or the vector expressing the IGF2 protein of the invention can (a) prevent and/or treat inflammatory diseases.

The IGF2 protein or a vector expressing IGF2 protein of the invention may also be used for one or more uses selected from the group consisting of:

(i) reducing pathogenic mononuclear cell infiltration in the colon;

(ii) inhibiting the pathogenic phenotype of macrophages;

(iii) reducing macrophage infiltration number;

(v) preventing and/or treating autoimmune diseases;

(vii) facilitating the implantation of the transplanted tissue or cells.

The invention also includes polypeptides or proteins having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, e.g., 99%) homology to the sequence shown in SEQ ID No. 1 of the invention and having the same or similar functions.

Wherein, SEQ ID NO. 1 is human IGF2 protein (AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE).

The "same or similar functions" mainly refer to: "(a) preventing and/or treating an inflammatory disease; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) facilitating the implantation of transplanted tissue or cells.

The protein of the invention can be recombinant protein, natural protein and synthetic protein. The proteins of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells). Depending on the host used in the recombinant production protocol, the protein of the invention may be glycosylated or may be non-glycosylated. The proteins of the invention may or may not also include an initial methionine residue.

The invention also includes fragments and analogs of the IGF2 protein that have IGF2 protein activity. As used herein, the terms "fragment" and "analog" refer to a protein that retains substantially the same biological function or activity of a native IGF2 protein of the invention.

In a preferred embodiment, the mutant of IGF2 protein of the invention is mutated at the tyrosine corresponding to position 27 of SEQ ID No. 1 (AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE) of wild-type IGF2 protein, and the mutant of IGF2 protein of the invention is significantly (a) preventive and/or therapeutic for inflammatory diseases; and/or (b) preventing and/or treating autoimmune disease.

Preferably, the tyrosine at position 27 is mutated to leucine.

In a preferred embodiment, the IGF2 mutant is a polypeptide having an amino acid sequence as set forth in any one of SEQ ID nos. 2-58, an active fragment thereof, or a conservatively variant polypeptide thereof.

It is to be understood that the amino acid numbering in the mutants of the invention is based on SEQ ID No. 1, and that when a mutant has 80% or more homology to the sequence shown in SEQ ID No. 1, the amino acid numbering of the mutant may be misaligned with respect to the amino acid numbering of SEQ ID No. 1), e.g. by 1-5 positions towards the N-terminus or C-terminus of the amino acid, whereas with sequence alignment techniques as are conventional in the art, it will generally be understood by those skilled in the art that such misalignment is within reasonable bounds and that it should not be possible to significantly (a) prevent and/or treat inflammatory diseases with the same or similar homology of 80% (e.g. 90%, 95%, 98%) due to the misalignment of the amino acid numbering; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) an activity to promote the implantation of transplanted tissue or cells is not within the scope of the present mutants.

The mutein fragment, derivative or analogue of the invention may be (i) a mutein wherein one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a mutein having a substituent group in one or more amino acid residues, or (iii) a mutein wherein the mature mutein is fused to another compound, such as a compound that extends the half-life of the mutein, e.g. polyethylene glycol, or (iv) a mutein wherein an additional amino acid sequence is fused to the mutein sequence, such as a leader or secretory sequence or a sequence used to purify the mutein or a proprotein sequence, or a fusion protein with an antigenic IgG fragment. Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein. In the present invention, conservatively substituted amino acids are preferably generated by amino acid substitutions according to Table I.

TABLE I

The present invention also includes polypeptides or proteins having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, e.g., 99%) homology to the native IGF2 protein of the present invention, which have the same or similar functions. The protein variant may be a derivative sequence obtained by substituting, deleting or adding at least one amino acid by several (usually 1 to 60, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and adding one or several (usually less than 20, preferably less than 10, more preferably less than 5) amino acids at the C-terminal and/or N-terminal. For example, in the protein, when the performance similar or similar amino acid substitution, usually does not change the protein function, C terminal and/or \ terminal addition of one or several amino acids usually does not change the protein function. The invention includes that the difference between the natural IGF2 protein analogue and the natural IGF2 protein can be difference in amino acid sequence, or difference in modified form which does not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other well-known biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein such as acetoxylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those performed during protein synthesis and processing. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). In addition, the mutant protein can be modified. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the mutein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the mutein or during further processing steps. Such modification may be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins which have been modified to increase their resistance to proteolysis or to optimize solubility.

The invention also provides polynucleotide sequences encoding IGF2 protein. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or artificially synthesized DNA, the DNA may be single-stranded or double-stranded. Polynucleotides encoding mature polypeptides include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences. The present invention also relates to variants of the above polynucleotides encoding fragments, analogs and derivatives of the polypeptides having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The coding nucleic acids of the invention can be readily prepared by one of skill in the art using a variety of known methods based on the nucleotide sequences described herein. Such methods are for example but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in sambrook, molecular cloning guidelines. As an embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences by segmentation and then performing overlap extension PCR.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) with the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more.

The proteins and polynucleotides of the invention are preferably provided in isolated form, more preferably, purified to homogeneity.

The full-length sequence of the polynucleotide of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

Methods for amplifying DNA/RNA using PCR techniques are preferably used to obtain the polynucleotides of the invention. Particularly, when it is difficult to obtain a full-length cDNA from a library, it is preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

Expression vector

The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells engineered with the vectors of the invention or the mutein-encoding sequences of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant muteins by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a mutein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the polynucleotide sequence encoding the mutein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the muteins of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, plant cells (e.g., ginseng cells).

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. Examples include the SV40 enhancer at the late side of the replication origin at 100 to 270 bp, the polyoma enhancer at the late side of the replication origin, and adenovirus enhancers.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

non-IGF 1R-binding substances

In the present invention, the non-IGF 1R-binding type substances include substances having selectivity or affinity for IGF2R higher than that of IGF1R, and substances which do not activate IGF1R (e.g., non-IGF 1R ligand such as retinoic acid) that can activate IGF2R with high efficiency. In a preferred embodiment, the non-IGF 1R-binding species has a higher affinity or selectivity for IGF2R than for IGF 1R. In a preferred embodiment, the non-IGF 1R-bound species is selected from the group consisting of: an IGF2 mutant, a vector expressing an IGF2 mutant, an antibody, a small molecule compound, or a combination thereof.

In a preferred embodiment, the non-IGF 1R-bound material further comprises one or more materials selected from the group consisting of: IGF2R, such as Plasminogen (Plasminogen), filaggrin (Serglycin), cell inhibitor of E1a stimulatory gene (CREG), Sulfamidase (Sgsh), Phosphorylated β -glucuronidase (GUSb), effectively inhibits inflammatory ligands.

In the present invention, it was surprisingly found for the first time that a substance other than IGF 1R-bound type could significantly (a) prevent and/or treat inflammatory diseases; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) facilitating the implantation of transplanted tissue or cells.

Compound pharmaceutical composition and medicine box

The present invention provides a composition comprising as an active ingredient (a) macrophages treated with a non-IGF 1R-binding form of the substance; (b) optionally a non-IGF 1R-binding species; and (c) a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for inhalation by nebulization. The pharmaceutical composition of the invention is in the form of injection. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation. The pharmaceutical composition of the present invention is preferably an injectable formulation. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents. Also, the pharmaceutical composition of the present invention may further comprise an additional component selected from the group consisting of: (a) a medicament for preventing and/or treating an inflammatory disease; and/or (b) a component for preventing and/or treating an autoimmune disease.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. Generally, the active ingredient of the present invention is used at a daily dose of about 0.01. mu.g to 1mg/kg body weight, preferably 0.1 to 10. mu.g/kg body weight, more preferably 0.5 to 5. mu.g/kg body weight. Can obtain satisfactory effect by the administration of the dosage. The optimum dosage will be adjusted as appropriate to the course of the disease and the condition being treated, for example, by the exigencies of the condition being treated, several divided doses may be administered daily, or the doses may be reduced proportionally.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

The present invention also provides a medicament useful for (a) preventing and/or treating inflammatory diseases; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon, and/or (ii) inhibiting the pathogenic phenotype of macrophages, and/or (iii) reducing the number of macrophage infiltrations, and/or (iv) conferring on macrophages an immune memory that significantly inhibits inflammation, and/or (v) preventing and/or treating autoimmune diseases, and/or (vi) treating a variety of other diseases in which macrophages are involved (such as inflammatory reactions associated with the development of diseases such as liver fibrosis, kidney fibrosis, lung fibrosis, etc., inflammatory reactions resulting from tissue, organ or cell transplantation, inflammatory reactions associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, lymphoma, etc., and inflammatory reactions associated with autoimmune diseases), and/or (vii) facilitating the implantation of transplanted tissues or cells, the kit comprises:

(i) a first container, and in the first container, an active ingredient (a) macrophage treated with a substance other than IGF 1R-bound form, or a medicament containing the active ingredient (a); and

(ii) instructions describing the administration of active ingredient (a) to (a) prevent and/or treat inflammatory diseases; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) instructions for facilitating the implantation of the transplanted tissue or cells.

The pharmaceutical compositions and kits of the invention are useful for (a) preventing and/or treating inflammatory diseases; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) facilitating the implantation of transplanted tissue or cells.

The formulations of the present invention may be administered three times per day to once every ten days, or once every ten days in a sustained release manner. The preferred mode is once a day because this facilitates patient adherence and significantly improves patient compliance with the medication.

When administered, the total daily dose to be administered in most cases will generally be lower (or equal to or slightly greater in a few cases) than the daily usual dose for each individual drug, although the effective dose of the active ingredient employed will vary depending on the mode of administration and the severity of the condition to be treated, etc.

Method of treatment

The invention also provides the use of two active ingredients of the invention or corresponding medicaments for (a) the prevention and/or treatment of inflammatory diseases; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) a method for promoting the engraftment of transplanted tissues or cells, which comprises administering to a mammal an effective amount of the active ingredient (a) macrophages having been treated with a substance other than IGF 1R-bound type, or a pharmaceutical composition containing said active ingredient (a); and (b) a substance other than IGF 1R-binding type, or a pharmaceutical composition containing the active ingredient (b).

When the two active ingredients of the present invention are used for the above-mentioned purpose, they may be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and may be orally administered in the form of: tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, from about 0.05 to 5% suspending agent), syrups (containing, for example, from about 10 to 50% sugar), and elixirs (containing, for example, from about 20 to 50% ethanol), or may be administered parenterally in the form of sterile injectable solutions or suspensions (containing from about 0.05 to 5% suspending agent in an isotonic medium). For example, these pharmaceutical preparations may contain from about 0.01% to about 99%, more preferably from about 0.1% to about 90%, by weight of the active ingredient in admixture with a carrier.

The two active ingredients or pharmaceutical compositions of the present invention may be administered by conventional routes including, but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral, or topical administration. Preferred routes of administration include oral, intramuscular or intravenous administration.

From the standpoint of ease of administration, the preferred pharmaceutical composition is a liquid composition, especially an injection.

In addition, the two active ingredients or the medicament of the present invention may be used in combination with other (a) agents for preventing and/or treating inflammatory diseases; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) a component or drug (nonsteroidal anti-inflammatory drug, glucocorticoid, methotrexate, TNF α neutralizing antibody, TNFR1 antibody, TNFR2 antibody, anti-CD 20 antibody, IL-1R antagonist, IL-12 and IL-23p40 neutralizing antibody, IL-23p19 neutralizing antibody, IL-17A receptor neutralizing antibody, CTLA-4 fusion protein) that promotes engraftment of transplanted tissue or cells.

The main advantages of the invention include:

(1) the invention discovers for the first time that a substance which is not IGF 1R-bound can significantly (a) prevent and/or treat inflammatory diseases; and/or (i) reducing pathogenic mononuclear cell infiltration in the colon; and/or (ii) inhibiting the pathogenic phenotype of macrophages; and/or (iii) reducing macrophage infiltration numbers; and/or (iv) immune memory conferring significant inhibition of inflammation to macrophages; and/or (v) the prevention and/or treatment of autoimmune diseases; and/or (vi) for a variety of other diseases in which macrophages are involved and which involve inflammatory responses (e.g., inflammatory responses associated with the development of diseases such as liver fibrosis, kidney fibrosis, and lung fibrosis, inflammatory responses associated with tissue, organ, or cell transplantation, inflammatory responses associated with the development and metastasis of cancers such as lung cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, melanoma, and lymphoma, and inflammatory responses associated with autoimmune diseases); and/or (vii) facilitating the implantation of transplanted tissue or cells.

(2) The present invention first found that insulin-like growth factor 2(IGF2) confers an immune memory that induces macrophage anti-inflammation. This property is dependent on the IGF2 receptor (IGF 2R). Functionally blocking IGF2R, IGF2 no longer confers the macrophage an immunological memory against inflammation.

(3) The invention discovers for the first time that the IGF2 with low dose can induce non-classical mitochondrial dynamics, endows macrophages with immune memory for resisting inflammation, and IGF2 with high dose can activate IGF1 receptor (IGF1R), inhibit the effect of IGF2R and promote monocytes and macrophages to obtain proinflammatory capacity by aerobic glycolytic metabolism.

(4) The invention discovers for the first time that IGF2 mutant with low affinity with IGF1R specifically activates IGF2R, and thus autoimmune diseases and inflammatory diseases such as peritonitis and inflammatory bowel disease are relieved to the greatest extent. These findings indicate that IGF2R targeted activation can determine the long-term immunosuppressive memory of macrophages, and IGF2R is an ideal target for treating inflammation-related diseases involving macrophages

(5) The invention discovers for the first time that IGF2R which directionally activates monocytes inhibits inflammatory bowel disease.

(6) The invention discovers for the first time that IGF2 is dependent on macrophages for treating inflammatory bowel disease.

(7) The invention discovers that IGF2 is involved in various other inflammatory reaction diseases of macrophage for the first time.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

All materials and reagents used in the examples are commercially available products unless otherwise specified.

Materials and methods

1 reagent

The research relates to a large number of experimental reagents, and in order to reduce the number of words and save the layout, the experimental reagents are listed in sequence according to different brands of the reagents.

Recombinant human insulin-like growth factor-2 (Recombinant human IGF2, 292-G2-250), Recombinant murine macrophage colony stimulating factor (Recombinant mouse M-CSF protein, 416-ML-050) was purchased from R & D Systems.

Human 27 leucine point mutant IGF2(Human Leu27-IGF2, TU100) was purchased from GroPep Bioreagens.

2 construction and identification of laboratory mice

IGF2R^+/-The mouse uses the Crisper-Cas9 technology to knock out 2bp or 13bp base on the 6 th exon of igf2r gene, resulting in frame shift mutation. Since IGF 2R-deficient homozygotes are embryonic lethality, we used IGF2R^+/-And (5) carrying out conservation, passage and identification on heterozygote mice. The primers used for identification are:

Primer 1,5’-CGTGAAGTCTGTCTATGGAAGAGACTGGACC-3’(SEQ ID NO.:59)，

Primer 2,5’-CATTACCAAGCTCACACTCCCTTCTCTTCTCTATT-3’(SEQ ID NO.:60),

the genotype result determination method will be embodied in the experimental results section.

Igf2r^fl/flMouse is characterized by that the loxp sequence is inserted into both sides of No. 2 exon of igf2r geneThe primers used were:

Primer 1,5’-CCCCGGTGTTGAGGTTGATAGATA-3’(SEQ ID NO.:61)，

Primer 2,5’-CAATTTAGGGCTGAAGCGATGGAT-3’(SEQ ID NO.:62),

Igf1r^fl/flPrimers used for mouse identification were:

Primer 1,5’-CTTCCCAGCTTGCTACTCTAGG-3’(SEQ ID NO.:63)，

Primer 2,5’-CAGGCTTGCAATGAGACATGGG-3’(SEQ ID NO.:64)，

Primer 3,5’-TGAGACGTAGCGAGATTGCTGTA-3’(SEQ ID NO.:65)

Lyz2^CrePrimers used for mouse identification were:

Primer 1,5’-CCCAGAAATGCCAGATTACG-3’(SEQ ID NO.:66)，

Primer 2,5’-CTTGGGCTGCCAGAATTTCTC-3’(SEQ ID NO.:67)，

Primer 3,5’-TTACAGTCGGCCAGGCTGAC-3’(SEQ ID NO.:68)

3 peritonitis mouse model

5% (mass/volume) of thioglycollate medium aqueous solution (5% Ftg) was prepared, sterilized at 121 ℃ under high temperature and high pressure for 30 minutes, and cooled to room temperature for use. Before use, physical properties of the thioglycollate culture medium are checked, the viscosity is preferably not to generate bubbles when the culture medium is shaken, and the color of the culture medium is preferably changed from blue-green color when initially prepared to brown when the culture medium is shaken.

C57/BL6 female mice of the same week age in the range of 9-12 weeks were selected for the experiments. Each mouse was slowly injected with 2ml of thioglycolate medium to the abdominal cavity, after 24 hours, IGF2 or other drug treatment was given, and the mice were euthanized at 72 hours after 24 hours and 48 hours after thioglycolate injection, and abdominal macrophages were isolated.

4 peritoneal macrophage isolation

The method comprises the steps of soaking euthanized mice with thioglycolate (Ftg) in 75% ethanol for several minutes, inducing the mice for 72 hours, tearing off abdominal fur along the direction perpendicular to the heads and the tails of the mice, sucking 10ml of Phosphate Buffer (PBS) precooled at four degrees centigrade by using an injector, wherein in experiments, the precooled PBS is used for irrigating the abdominal cavities of the mice to obtain the maximum number of cells, forcefully and quickly injecting the phosphate buffer (helping to blow down adherent cells) into the abdominal cavities of the mice, clamping the tails of the mice by the index finger and the ring finger of the right hand, lightly holding the mice, shaking the mice up and down violently for three to five times, and slowly sucking out cell lavage liquid by using the injector. The peritoneal macrophage lavage fluid needs to be stored for four degrees, and cells are easy to adsorb on the tube wall of the centrifugal tube at normal temperature.

5 inflammatory bowel disease model

A16% (mass/volume) aqueous dextran sulfate sodium salt (DSS) solution was prepared, sterilized by filtration through a 0.45 μ M frit, and passed into an SPF barrier. A 4-fold dilution to a 4% final concentration was performed on a 16% DSS solution in regular drinking water. Female mice of 9-12 weeks of age were selected to be fed with 4% DSS solution for different days, with the expiration date varying according to the purpose of the experiment. In experiments in which IGF2 therapeutic efficacy was initially demonstrated, 4% DSS solution was fed continuously and terminated after completion of survival curve statistics; the subsequent experiments were usually fed continuously with 5 days of 4% DSS solution, and the specific drinking times for the different batches of experiments will be reflected in the experimental results.

Clinical scoring for inflammatory bowel disease was performed according to the following criteria. "Stool scenes" and "dying scenes" were evaluated 4 days after mice were fed with 4% DSS solution. The "stock scores" criteria are: 0 minute, the shape of the excrement is normal; 1 minute, semi-formed feces and no attachment of anus; 2 min, semi-formed feces and anus adhesion exist; and 3 min, the liquid feces are attached to the anus. "assessment criteria for the blowing categories": 0 point, negative fecal occult blood test paper; 1 point, positive fecal occult blood test paper; 2 min, blood marks can be seen in the cages; 3 points, bleeding in the rectum and red blood spots in the anus.

6 colonic infiltrating lymphocyte isolation

Mice were euthanized by 4% DSS induction for 4-5 days, dissected, and colonic tissue was obtained, placed in four degrees celsius pre-cooled phosphate buffer, and the surface of the colon was washed free of blood. The colon was cut open longitudinally and the feces carefully washed away in a four-degree precooled phosphate buffer. Adding DTT into 1640 culture solution containing 200 mu.g/ml DNaseI and 1mg/ml type VIII collagenase before digestion, digesting for 60 minutes by using a shaker at 200 rpm, carefully taking out colon basement layer tissues, cutting into small segments, re-digesting, filtering by using a 70 mu m cell screen, and centrifuging the filtrate for 5 minutes at the rotating speed of 400g to obtain a single cell precipitate.

Preparing 40% and 80% Percoll separating medium, resuspending single cell pellet with 2ml volume of 40% Percoll, carefully adding 80% Percoll separating medium to the bottom of the centrifuge tube at the lower layer of the 40% Percoll, and properly controlling the pressure on the pipette to prevent the two layers of separating medium from mixing and becoming turbid. Adjusting the temperature of the centrifuge to 25 ℃, enabling the ascending and descending acceleration gradient to be 0, and centrifuging for 25 minutes at 2000 rpm. The layer cells in the middle of the 40% and 80% Percoll isolates were collected and washed at least twice with 10ml of four-c pre-cooled phosphate buffer to completely remove Percoll, and pure colon-derived mononuclear cells were finally obtained.

7 flow cytometry analysis

Since monocytes and macrophages express the receptor for IgG, CD16/32, at relatively high levels, for staining of the monocyte and macrophage antibodies, the cells need to be blocked in advance with fluorescein-free conjugated CD16/32 antibody (antibody: phosphate buffered saline ═ 1: 50 ratio diluted antibody, 100 μ l antibody working solution/1 × 10)⁷Cells, incubated for 30 minutes at room temperature). Because macrophages have the characteristics of being adherent and non-digestible, a culture plate with ultra-low adsorption should be selected for bearing cells when the macrophages are stained.

Staining cell membrane surface proteins. For CD16/32 antibody-blocked cells, the ratio was 1: 100 proportions of diluted antibody with phosphate buffer, 100. mu.l staining broth/1X 10⁷Cells were stained at room temperature for 30 minutes, washed with phosphate buffer, centrifugedResuspend with 200. mu.l phosphate buffer, mount to test or fix with 1% neutral formaldehyde.

Intracellular and intracellular protein staining. Intracellular and nuclear proteins need to be carried out after the protein staining on the cell membrane surface is completed. For intracellular proteins, membrane disruption and post-Fixation staining were performed using a cell Fixation and membrane disruption kit (Fixation/Permeabilization Solution and Perm/Wash Buffer); for the nuclear proteins, cell fixation/cell rupture kit (Foxp 3/Transcription Factor stabilizing Buffer Set) was used for fixation and post rupture Staining.

8 extracellular flux analysis (Hippocampus japonicus experiment)

The culture medium of peritoneal macrophages or THP1 cells was replaced with Seahorse's special medium (XF medium) supplemented with 10mM glucose, 2mM glutamine and 2mM pyruvate. Oxygen consumption rate determination experiments and extracellular acid production rate determination experiments with or without the following stimuli: mu.M oligomycin, 0.75mM FCCP, 100nM rotenone and 1. mu.M antimycin were assayed using an extracellular flow analyzer (Agilent).

9 real-time quantitative PCR

Total cellular RNA was extracted using a Tiangen biological cell/bacterial RNA isolation kit, and a volume of 300ng/ml RNA of 30. mu.L per sample was typically obtained. All RNAs were reverse transcribed into cDNA using the Takara reverse transcription kit (PCR conditions: 37 ℃ for 15 minutes, 85 ℃ inactivation for 5 seconds, temperature drop to 4 ℃ for storage). The cDNA can be stored for several days at minus twenty degrees centigrade, and can be degraded in a long time.

Before using the cDNA, the cDNA was diluted 10-fold with double distilled water and the real-time quantitative PCR system was 10. mu.L, containing 5. mu.L SYBR, 4. mu.L cDNA, 1. mu.L primer. After spotting the samples in 384 well plates, centrifugation was carried out at 2500 rpm for 2 minutes at room temperature, which would result in a water mist on the membrane if centrifuged at 4 ℃. The real-time quantitative PCR conditions were:

step

1, 95 ℃ for 10 min,

Step

2, 95 ℃ for 15 sec,

Step

3, 60 ℃ for 1 min,

Step

4, 95 ℃ for 15 sec,

Step

5, 60 ℃ for 1 min,

Step

6, 95 ℃ for 15 sec, wherein Step 2 and Step 3 were cycled 40 times.

The primers involved in the real-time quantitative PCR experiment were as follows:

TNFα-F：5’-CCTGTAGCCCACGTCGTAG-3’(SEQ ID NO.:69)，

TNFα-R：5’-GGGAGTAGACAAGGTACAACCC-3’(SEQ ID NO.:70)；

IL-1β-F：5’-GCAACTGTTCCTGAACTCAACT-3’(SEQ ID NO.:71)，

IL-1β-R：5’-ATCTTTTGGGGTCCGTCAACT-3’(SEQ ID NO.:72)；

Cxcl10-F：5’-CCAAGTGCTGCCGTCATTTTC-3’(SEQ ID NO.:73)，

Cxcl10-R：5’-GGCTCGCAGGGATGATTTCAA-3’(SEQ ID NO.:74)；

Retnla-F：5’-CCAATCCAGCTAACTATCCCTCC-3’(SEQ ID NO.:75)，

Retnla-R：5’-ACCCAGTAGCAGTCATCCCA-3’(SEQ ID NO.:76)；

CCL22-F：5’-AGGTCCCTATGGTGCCAATGT-3’(SEQ ID NO.:77)，

CCL22-R：5’-CGGCAGGATTTTGAGGTCCA-3’(SEQ ID NO.:78)；

Clec7a-F：5’-GACTTCAGCACTCAAGACATCC-3’(SEQ ID NO.:79)，

Clec7a-R：5’-TTGTGTCGCCAAAATGCTAGG-3’(SEQ ID NO.:80)；

10H & E staining

Dehydrating and embedding: the colon tissue at the same position is fixed in 4% paraformaldehyde solution for 24 hours, dehydrated and embedded sequentially through the following steps. Soaking in 70% ethanol overnight; soaking in 80% ethanol for 2 hr; soaking in 85% ethanol for 1.5 hr; soaking in 90% ethanol for 1 hr; soaking in 95% ethanol for 1 hr; soaking in 100% ethanol for 30 min, and repeating the above steps; soaking the mixture in xylene for 5 minutes, and repeating the operation once; soaking the liquid paraffin at the temperature of 60 ℃ for 1 hour, and repeating the operation once; paraffin embedding was accomplished using a paraffin embedding machine.

And (3) staining the section: sections were sliced at 3 μm thickness, paraffin tissue sections were stretched in a 56 ℃ water bath for several seconds, adsorbed onto a glass slide, oven dried overnight at 60 ℃ and then stained as follows. Soaking the mixture in xylene for 5 minutes, and repeating the operation once; soaking in 100% ethanol for 5 min, and repeating the above steps; soaking in 95% ethanol for 5 min, and repeating the above steps; soaking in 70% ethanol for 5 min; distilled water, soaking for 5 minutes; hematoxylin, and staining for 5 minutes; flushing running water for a proper time; ammonia water, and soaking and washing for a proper time; flushing for a proper time by running water; eosin, staining for 1 minute; soaking in 95% ethanol for 1 min; soaking in 100% ethanol for 1 min, and repeating the above steps. Soaking in xylene for 1 min; repeating the operation once; the sealing agent seals the sheet.

11 immunohistochemistry

Paraffin section antigen renaturation: drying the paraffin sections in a drying oven at 60 ℃ for two hours; washing the paraffin sections three times with phosphate buffer solution with pH 7.4 for three minutes each time; placing the deparaffinized, hydrated paraffin sections in boiling citrate buffer at pH 6.0 for ten minutes; after running water is naturally cooled, the mixture is washed twice with distilled water for three minutes each time; the cells were washed twice with three minutes each time in phosphate buffer pH 7.4. One drop of 3% hydrogen peroxide was added to each paraffin section and incubated for ten minutes at room temperature.

Antigen-antibody reaction: dripping primary antibody of anti Ki67 on the paraffin section, and incubating overnight at four ℃; four rinses with phosphate-tween buffer pH 7.4 for three minutes each; dripping a horseradish peroxidase-coupled secondary antibody on the paraffin section, and incubating for sixty minutes at room temperature; four rinses with phosphate-tween buffer pH 7.4 for three minutes each; after DBA dyeing is carried out for a proper time, washing away the DBA dyeing agent; counterstaining the paraffin sections by using hematoxylin dye, and determining proper time according to the tissue type; washing with tap water to remove lignum sappan threo; using hydrochloric acid alcohol for differentiation and ammonia water for anti-blue, until the hematoxylin and perilla staining presents light blue; and (5) dehydrating and sealing.

TUNEL immunohistochemical experiments were performed according to the experimental procedure provided by In situ cell death detection kit-POD kit (Roche, TUN 11684817).

12 nitric oxide detection

50 μ L of LPS-stimulated 24-hour macrophage culture medium was pipetted into 96-well flat-bottom clear plates, eachThis sets up 3 auxiliary holes. Adding 50 mu L of Griess reagent into each well, mixing uniformly, incubating for 15 minutes at room temperature in a dark place, and detecting the light absorption value under the wavelength of 540nm of a microplate reader. For the quantitative determination experiment, 100mM NaNO can be prepared in advance₂The standard mother liquor, the standard with different concentrations diluted in gradient of 0, 5, 10, 20, 30, 40, 50, 100. mu.M, is added with equal volume of Griess reagent, and standard curve is drawn after incubation and plate reading under the same conditions. The detection of the accumulation amount of the nitric oxide in the culture medium supernatant can reflect the activity of macrophage Inducible nitric oxide synthase (iNOS), and can predict the energy metabolism type and the inflammation phenotype of the macrophage.

13 lactic acid detection

Lactate production capacity of peritoneal monocytes and macrophages pre-programmed with IGF2 was tested using a lactate detection kit. The experiments were performed according to the experimental method provided by the Lactate Colorimetric Assay Kit II (BioVision, K627-100). And detecting the accumulation amount of lactic acid in the culture medium supernatant, wherein the accumulation amount can reflect the energy metabolism type and the inflammation phenotype of the macrophage.

14 statistical analysis of data

Data are presented as means or mean ± s.e.m. or s.d. using Graphpad software mapping and significant difference analysis, in this study the significance of the difference between the two sets of data was analyzed using the two-tailed nonparametric Student's t test. ns, meaning no significant difference; p < 0.05; p < 0.01; p <0.001, P <0.05 was considered to be significantly different.

Example 1 low dose IGF2 inhibited peritonitis.

Peritonitis mice were treated with low-dose IGF2(L-IGF2, 5-50ng IGF2 per mouse) and high-dose IGF2(H-IGF2, 1000ng IGF2 per mouse), respectively. Peritoneal monocytes and macrophages were isolated and analyzed from peritonitis mice.

The results show that low-dose IGF2 can significantly inhibit macrophage infiltration in the abdominal cavity of peritonitis mice (fig. 1) compared to the control group and the high-dose IGF 2-treated group, indicating that low-dose IGF2 can inhibit inflammatory conditions in peritonitis mice.

Example 2 anti-inflammatory macrophages under the control of low dose IGF2 predominate in the metabolic bias of oxidative phosphorylation.

Peritonitis mice were treated with low dose IGF2(L-IGF2, 5-50ng IGF2 per mouse) and high dose IGF2(H-IGF2, 1000ng IGF2 per mouse), respectively, peritonitis mice were isolated from peritoneal macrophages and analyzed for cellular metabolic status. The hippocampal experiment (Seahorse technology) is a gold standard to study the preference of cellular energy metabolism. We found from studies of hippocampal experiments that the maximum Oxygen Consumption Rate (OCR) of peritoneal macrophages pre-programmed with low dose IGF2 was significantly higher than that of the control group, and that the maximum Oxygen consumption rate of peritoneal macrophages pre-programmed with high dose IGF2 was lower than that of the control group (fig. 2A). Therefore, the low dose of IGF2 increased the oxygen consumption potential of peritoneal macrophages, while the high dose of IGF2 reversed this potential.

In addition, we also determined the maximum Extracellular acidification rate (ECAR) of these three groups of cells using the hippocampal experiment and found that the Extracellular maximum acid production rate of the peritoneal macrophages preprogrammed with low dose IGF2 was significantly lower than that of the PBS control group and the high dose IGF2 group. These results are better represented by the ratio of the maximum oxygen consumption rate to the extracellular maximum acid production rate (OCR: ECAR) (FIG. 2B). Low dose IGF2 pre-programmed macrophages exhibit higher levels of OCR: ECAR values indicate that this group of macrophages tend to acquire energy in an Oxidative phosphorylation (OXPHOSPHOS) predominant manner, whereas the macrophages in the control and high IGF2 groups tend to select the aerobic glycolysis for energy production required by the cells.

Lactic acid is an intermediate metabolite of aerobic glycolysis, and during the aerobic glycolysis, excessive lactic acid is secreted out of cells by cells, so that detecting and comparing the accumulation amount of lactic acid in the cell culture supernatant is an effective way to measure the aerobic glycolysis level of cells. We re-stimulated these isolated peritoneal macrophages with Lipopolysaccharide (LPS) and examined the lactic acid content in the culture supernatants after 24 hours, and found that the lactic acid accumulation in the culture supernatants of peritoneal macrophages pre-programmed with PBS and high dose IGF2 was significantly higher than in the low dose IGF2 treated group (fig. 2C). The above studies indicate that low doses of IGF2 confer stronger oxidative phosphorylation capacitation potential to mouse peritoneal macrophages, while high doses of IGF2 reverse this property and even increase the anaerobic glycolytic capacitation preference of cells. Macrophages that have oxidative phosphorylation as the primary capacitation often have the characteristic of replacing activated macrophages, representing a phenotype of resisting inflammation. To validate the immunophenotype of peritoneal macrophages pre-programmed with different doses of IGF2, it was investigated to treat these isolated macrophages with Lipopolysaccharide (LPS) and interleukin-4/13 (IL-4/13), respectively, mimicking both the classical and alternative activation pathways to stimulate macrophages. Real-time quantitative PCR (RT-PCR) results showed that inflammatory factor expression of lipopolysaccharide classically activated macrophages was inhibited by low dose IGF2(L-IGF2), and high dose IGF2 was not effective for part of the genes (fig. 2D); the anti-inflammatory factor expression of interleukin 4/13 replacement activated macrophages was amplified by IGF2(L-IGF2) and high dose IGF2 was not effective (fig. 2E, F).

These results demonstrate that the oxidative phosphorylation energy metabolism preference induced by low-dose IGF2 promotes a strong anti-inflammatory phenotype in peritoneal macrophages, while the high-dose IGF2 reverses the effects of low-dose IGF2, reverting the energy metabolism pattern to an aerobic glycolysis-dominated pattern close to that of the control group, and correspondingly reverses the inflammatory phenotype.

Example 3

Igf2r gene deficient mice, as well as igf1r or igf2r conditional knockout mice were constructed.

IGF2 binds to IGF1R and IGF2R with the greatest affinity for IGF2R, and when IGF2 binds to IGF2R, leading to internalization of IGF2R, IGF2 will be transported to lysosomes for degradation, so IGF2R is often considered to be a Decoy receptor (Decoy receptor) for IGF2, whereas IGF2 binds to IGF1R and performs mitogen function, promoting cell proliferation and survival². Based on the previous section, we found that high doses of IGF2 demonstrated mitogen function and promoted proliferation and survival of peritoneal macrophages in peritonitis mice, whereas low doses of IGF2 did not. In further view of the maximal affinity of IGF2 for IGF2R, we hypothesized that low-dose IGF2 preferentially binds to IGF2R, inducing IGF2R-dependent biological effects, such as the onset of non-classical mitochondrial kinetics, high doses of IGF2 remain available to bind IGF1R after "depleting" the extracellular membrane IGF2R, counteracting the biological effects of IGF2R, and promoting proliferation and survival of peritoneal macrophages in peritonitis mice.

To investigate which receptor IGF2 depends on, respectively, to achieve bidirectional regulatory function on monocytes, we constructed genetically engineered mice. In igf2r gene knockout mice, we targeted knockout of the igf2 gene at exon 6 base 2bp or 13bp using the cripper-Cas 9 technique, resulting in a frameshift mutation (fig. 3A). Since complete deletion of IGF2r gene can cause embryonic lethality in mice, we finally obtained IGF2r knockout heterozygote mice, IGF2R^+/-Mice were bred, breed preserved and tested, IGF2R^+/-Mouse progeny genotypic identification was dependent on sequencing alignment (fig. 3B). We induced mouse peritoneal macrophages and confirmed the knockout efficiency by flow-testing the expression of IGF2R in these cells (fig. 3C).

To construct IGF2r conditional knockout mice, we first constructed IGF2R by introducing loxp sequences in the same direction at both ends of the second exon of IGF2r gene using ES cell targeting technology^fl/flA mouse. Then IGF1R was added^fl/flMouse and IGF2R^fl/flMice were separately administered with Lyz2^CreMice are mated, where Lyz2 is expressed at high levels in myeloid lymphocytes¹⁸Until IGF1R is obtained^fl/flLyz2^CreHomozygote mouse and IGF2R^fl/flLyz2^CreHomozygous mice (fig. 3D, G). The results of DNA-level and protein-level identification confirmed the knock-out efficiency (fig. 3E, F, H, I).

Example 4 targeted monocyte activation of IGF2R inhibits inflammatory bowel disease.

To evaluate the role of IGF2-IGF2R preprogrammed monocytes and macrophages in other inflammatory models, we induced an inflammatory bowel disease model using dextran sulfate sodium salt (DSS), an experimental animal model most similar to the symptoms of human crohn's disease and ulcerative colitis^19,20. Next, we used different agentsAmounts of IGF2 and IGF 2-treated peritoneal macrophages were treated for DSS-induced inflammatory bowel disease.

4.1 Low dose IGF2 inhibits DSS-induced inflammatory bowel disease

We found that low dose IGF2 treatment significantly slowed the weight loss and significantly prolonged the survival time of mice induced by DSS, whereas high dose IGF2 accelerated the weight loss and failed to prolong the survival time of mice (fig. 4A, B). In addition, we also recorded and evaluated other indices of mice, such as "Stool Score" and "Bleeding Score" and colon length, and found that low dose IGF2 could significantly reduce the scores of "Stool Score" and "Bleeding Score" and better preserve the length of mouse colon (fig. 4C, D, E).

In the H & E pathology results, we found that the colon villi were mostly destroyed in the control and high-dose IGF 2-treated mice, while the low-dose IGF2 group was relatively intact (fig. 4F). In the TUNEL immunohistochemical staining results, we found that dead cells spread along the colonic villi to the basal layer, and that the positive death signals were significantly stronger in the control and high dose IGF 2-treated groups than in the low dose IGF 2-treated group (fig. 4G). In the results of Ki67 immunohistochemical staining, it was found that, as the Ki67 positive proliferation signal extends from the basement membrane to the direction of villus, the Ki67 positive signal of the low-dose IGF2 group is significantly stronger than those of the control group and the high-dose IGF2 treatment group, which indicates that monocytes and macrophages near the basement membrane play a role in the repair of intestinal villus, and macrophages at the colon basement membrane of the mouse treated with the low-dose IGF2 can better promote the repair of the colon villus (fig. 4H).

In conclusion, the low dose of IGF2 could significantly improve the quality of life and survival of DSS-induced inflammatory bowel disease mice, while the high dose of IGF2 had no therapeutic effect and even aggravated some symptoms.

4.2IGF2 treatment of inflammatory bowel disease depends on macrophages

To verify the key role of IGF2 pre-programmed monocytes and macrophages in DSS-induced inflammatory bowel disease, we treated DSS-induced inflammatory bowel disease mice with PBS, low dose IGF2, and high dose IGF2 pre-programmed peritoneal macrophages. As a result, it was found that the weight loss and survival time of the inflammatory bowel disease mice treated with PBS-pretreated and high-dose IGF 2-pretreated peritoneal macrophages was increased, while the body mass index and survival time of the inflammatory bowel disease mice treated with low-dose IGF 2-pretreated peritoneal macrophages were significantly improved (fig. 5A, B).

Therefore, macrophages play a crucial role in both DSS-induced inflammatory bowel disease that is alleviated by low doses of IGF2, and inflammatory bowel disease that is exacerbated by high doses of IGF 2.

4.3 Low dose IGF2 reduces pathogenic macrophage infiltration in the colon

In view of the important role played by macrophages in DSS-induced inflammatory bowel disease, we performed numerical and phenotypic analysis of colon-infiltrated mononuclear cells. The results indicate that low dose IGF2 treatment significantly reduced mononuclear cell infiltration in the colon, while high dose IGF2 increased mononuclear cell infiltration in colon tissue (fig. 6A). This phenomenon was similar to the number of peritoneal macrophages available in peritonitis mice treated with low-dose IGF2 and high-dose IGF 2.

Before testing the phenotype of isolated mononuclear cells in colon tissue from mice with inflammatory bowel disease, we stimulated these cells with lipopolysaccharide for 5 hours and added Brefeldin a (BFA) to inhibit intracellular factor release. The results of the experiment showed that macrophages in colon tissue of mice with inflammatory bowel disease treated with low dose of IGF2 expressed less interleukin-1 β (IL-1 β) relative to cells in the control group, whereas macrophages in colon tissue of mice with colitis treated with high dose of IGF2 expressed interleukin-1 β significantly higher (fig. 6B). At the same time we counted the proportion of pathogenic IL-1 β positive macrophages in the total infiltrated macrophages (FIG. 6C).

These results indicate that low dose IGF2 treatment inhibited the pathogenic phenotype of macrophages infiltrated by colonic tissue in mice with inflammatory bowel disease while decreasing the number of macrophage infiltrates, whereas high dose IGF2 treatment exacerbated the pathogenic phenotype of macrophages while increasing the number of infiltrates in this population of cells.

4.4IGF2R performs the anti-inflammatory task of low-dose IGF2

To verify whether the mechanism of action of IGF2 in the treatment of DSS-induced inflammatory bowel disease mice is consistent with our previous findings, we generated inflammatory bowel disease in mice with DSS-induced myeloid lymphocyte-specific knock-out of IGF2R or IGF1R and treated with IGF 2. The results show that IGF2R was compared to the control group^fl/flMouse IGF2R with IGF2R specifically knocked out^fl/flLyz2^CreMice were more sensitive to DSS, weight loss was more pronounced, and low dose IGF2 no longer exerted a potent effect against inflammatory bowel disease, with a sustained loss in weight (fig. 7A).

Correspondingly, IGF1R was compared to control^fl/flMouse IGF1R with IGF1R specifically knocked out^fl/ ^flLyz2^CreMice were more resistant to DSS, and body weights of mice were significantly higher than IGF1R in the control group, both in the PBS-treated group and the high-dose IGF 2-treated group^fl/flBody weight of mice under the corresponding treatment conditions (fig. 7B).

The above experimental results show that in a DSS-induced mouse model of inflammatory bowel disease, treatment with IGF2 still relies on IGF2R to perform the modeling of the anti-inflammatory phenotype, while signaling of IGF1R activation promotes the pro-inflammatory phenotype of macrophages in inflammatory bowel disease.

4.5Leu27-IGF2 Targeted activation of IGF2R is most effective in inhibiting peritonitis and inflammatory bowel disease

In order to facilitate studies on the functions of IGF2R and IGF1R, a number of IGF2 mutants have been designed and reported, including IGF2 mutants that selectively bind IGF 2R. Researchers mutate tyrosine 27 of IGF2 peptide segment into leucine to prepare Leu27-IGF2 mutant (L27-IGF2), and Leu27-IGF2 mutant has unchanged affinity with IGF2R and IGFBP protein and extremely reduced affinity with IGF 1R.

Compared with wild-type IGF2, Leu27-IGF2 significantly decreased cytoplasmic relative pH of peritoneal monocytes and peritoneal macrophages in peritonitis mice, and even at high doses of 2000ng/ml, we compared the effect of Leu27-IGF2 on the change in cytoplasmic pH at low doses, eliminating the possibility of low Leu27-IGF2 titers (FIG. 8A). In response, peritoneal macrophages from peritonitis mice, pre-programmed with low-dose Leu27-IGF2 and high-dose Leu27-IGF2, both significantly inhibited the production of nitric oxide (fig. 8B) and lactic acid (fig. 8C) following lipopolysaccharide challenge. This indicates that both low and high dose Leu27-IGF2 and Leu27-IGF2 confer anti-inflammatory phenotype on peritoneal macrophages in peritonitis mice.

Similarly, both the low dose Leu27-IGF2 and the high dose Leu27-IGF2 significantly inhibited DSS-induced weight loss in the mouse model of DSS-induced inflammatory bowel disease (fig. 8D). The therapeutic effect of low and high dose Leu27-IGF2 and Leu27-IGF2 was also reflected in the length of the colon in these mice (fig. 8E).

From the above experimental results, it can be analyzed that the threshold for effective inflammation inhibition of Leu27-IGF2 was increased by more than 20-fold compared to wild-type IGF2 in both thioglycolate medium-induced peritonitis and DSS-induced inflammatory bowel disease models (fig. 8F).

In this section of the study, we validated the therapeutic role of IGF2 in a mouse model of DSS-induced inflammatory bowel disease. It was confirmed that the effective inhibition of inflammatory bowel disease by low-dose IGF2 relies on the same mechanism of action as the treatment of peritonitis by low-dose IGF2, and in both validated models, IGF 2R-dependent pathways can confer significant immune memory to macrophages that inhibits inflammation. The IGF2 mutant targets and activates IGF2R, exerting the most stable inflammation-inhibiting effect.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

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Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

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<210> 43

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 43

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Ser Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Leu Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 44

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 44

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Leu Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 45

<211> 67

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 45

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Asp Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala

50 55 60

Lys Ser Glu

65

<210> 46

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 46

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Asp Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 47

<211> 67

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 47

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Ala Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala

50 55 60

Lys Ser Glu

65

<210> 48

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 48

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Ala Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 49

<211> 67

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 49

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Gln Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala

50 55 60

Lys Ser Glu

65

<210> 50

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 50

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Gln Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 51

<211> 67

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 51

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile His Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala

50 55 60

Lys Ser Glu

65

<210> 52

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 52

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile His Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 53

<211> 67

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 53

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Arg Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala

50 55 60

Lys Ser Glu

65

<210> 54

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 54

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Arg Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 55

<211> 67

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 55

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Lys Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala

50 55 60

Lys Ser Glu

65

<210> 56

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 56

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Lys Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 57

<211> 67

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 57

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Leu Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Leu Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala

50 55 60

Lys Ser Glu

65

<210> 58

<211> 61

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 58

Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr

1 5 10 15

Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Leu Phe Ser Arg Pro Ala

20 25 30

Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Leu Glu Glu Cys Cys Phe

35 40 45

Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala

50 55 60

<210> 59

<211> 31

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 59

cgtgaagtct gtctatggaa gagactggac c 31

<210> 60

<211> 35

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 60

cattaccaag ctcacactcc cttctcttct ctatt 35

<210> 61

<211> 24

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 61

ccccggtgtt gaggttgata gata 24

<210> 62

<211> 24

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 62

caatttaggg ctgaagcgat ggat 24

<210> 63

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 63

cttcccagct tgctactcta gg 22

<210> 64

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 64

caggcttgca atgagacatg gg 22

<210> 65

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 65

tgagacgtag cgagattgct gta 23

<210> 66

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 66

cccagaaatg ccagattacg 20

<210> 67

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 67

cttgggctgc cagaatttct c 21

<210> 68

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 68

ttacagtcgg ccaggctgac 20

<210> 69

<211> 19

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 69

cctgtagccc acgtcgtag 19

<210> 70

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 70

gggagtagac aaggtacaac cc 22

<210> 71

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 71

gcaactgttc ctgaactcaa ct 22

<210> 72

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 72

atcttttggg gtccgtcaac t 21

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 73

ccaagtgctg ccgtcatttt c 21

<210> 74

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 74

ggctcgcagg gatgatttca a 21

<210> 75

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 75

ccaatccagc taactatccc tcc 23

<210> 76

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 76

acccagtagc agtcatccca 20

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 77

aggtccctat ggtgccaatg t 21

<210> 78

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 78

cggcaggatt ttgaggtcca 20

<210> 79

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 79

gacttcagca ctcaagacat cc 22

<210> 80

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 80

ttgtgtcgcc aaaatgctag g 21

Claims

1. Use of a substance which is not IGF 1R-bound, for the preparation of a composition or formulation for the prevention and/or treatment of an inflammatory disease.

2. The use according to claim 1, wherein the non-IGF 1R-bound substance is selected from the group consisting of: an IGF2 mutant, a vector expressing an IGF2 mutant, an antibody, a small molecule compound, or a combination thereof.

3. A cell preparation, comprising:

macrophages treated with a non-IGF 1R-binding agent.

4. A kit, comprising:

5. A method of screening pharmaceuticals, comprising the steps of:

6. A method of screening pharmaceuticals, comprising:

7. A method of obtaining a pro-inflammatory sample and an anti-inflammatory sample in vitro, comprising:

8. A composition, comprising:

(a) a mutant IGF2 that is not IGF 1R-bound obtained by the method of claim 5; wherein the non-IGF 1R-binding IGF2 mutant does not comprise an IGF2 mutant selected from the group consisting of: wild-type IGF2, 25-91 amino acid fragments of wild-type IGF2, IGF2 mutant in which glutamic acid at position 12 is mutated to Asp, Ala, Gln, His, Arg, or Lys, IGF2 mutant in which phenylalanine at position 26 is mutated to Ser, IGF2 mutant in which tyrosine at position 27 is mutated to Leu, IGF2 mutant in which valine at position 43 is mutated to Leu, IGF2 mutant in which D-domain is deleted, or a combination thereof.

9. A method of inhibiting inflammatory activity comprising the steps of:

10. Use of a cell preparation according to claim 3, a kit according to claim 4 or a composition according to claim 8 for the preparation of a medicament for the prophylaxis and/or treatment of inflammatory diseases.